Gamma-secretase is an unusual aspartyl protease that cleaves substrates within the transmembrane (TM) region. The presenilins (PS 1 and PS2) are essential for g-secretase-mediated cleavage of select TM proteins, including amyloid b-protein precursor (APP) to yield amyloid b-peptide (Ab). Familial Alzheimer's disease (FAD)-associated mutations in the presenilins give rise to an increased production of a longer and more amyloidogenic form of Ab (Ab42). In addition to their proteolytic activity, we reported that the functional presenilins are required to modulate the pathway for capacitative calcium entry (CCE), the refilling mechanism for depleted internal Ca2+ store. Furthermore, presenilin FAD mutations universally attenuate CCE. "Conformational coupling" mediated by direct physical interaction between plasma membrane CCE channels and ER constituents is known to be an underlying mechanism for CCE. Our studies suggest that Ca2+-mediated presenilin function and presenilin-associated g-secretase activity are functionally coupled and may be governed by a shared regulatory mechanism involving conformational coupling. We plan to study the underlying mechanism as to how the presenilins regulate Ca2+ entry and g-secretase-like proteolytic activity and what is the temporal and spatial relationship between these two separate presenilin functions (e.g. Ca2+-related vs. proteolytic). Additionally, we will identify relevant molecular effectors and substrates for these pathways. Our two central hypotheses are: i) the presenilin-associated g-secretase cleaves a substrate that is critical for calcium entry; ii) alternatively, the presenilins directly regulate "conformational coupling" between plasma membrane and ER, simultaneously serving as a regulatory mechanism for g-secretase and Ca2+ entry (e.g. CCE). To address these hypotheses, we will carry out three aims: 1) To define the precise relationship of Ca2+-related presenilin function with presenilin-associated g-secretase activity; 2) To determine the molecular nature and subcellular locus for the proteolytic and Ca2+-related functions of the presenilins; 3) To identify molecular substrates for presenilin-associated g-secretase. Our studies should provide information relevant in general to the integrated mechanism of Ca2+ mobilization and the proteolysis of TM proteins, and in particular to the underlying mechanism of FAD-associated neuropathogenesis.